Multiple Sclerosis (MS) is an inflammatory, demyelinating disorder of the central nervous system (CNS). The etiology of MS remains unclear, but the disease develops in genetically susceptible individuals exposed to environmental triggers. The long favored hypothesis in MS implicates autoreactive T cells generated in the periphery that access the CNS, where they induce injury of previously normal neural tissues. However, in contrast to the animal model experimental autoimmune encephalomyelitis (EAE), neither the target(s) of the immune response nor the cells responsible for CNS damage have been identified in MS. Furthermore, the failure of some MS treatments targeting processes that underlie the development of CNS tissue destruction in EAE (e.g. IFN-g, TNF-a inhibitors) indicates that different mechanisms may cause the development of disability in MS versus EAE. Therefore, there is a need to identify pathophysiological mechanisms that are specific for MS, but may not be predicted from EAE. Therapeutic trials, especially those that investigate novel therapeutic agents, represent a unique opportunity to investigate how specific perturbations of the biological system affect MS disease process. The goal of this project is to carefully study the biological perturbations induced by the application of novel therapeutic agents in Phase I/II clinical trials in MS in order to define mechanisms of CNS tissue injury, but also those that underlie beneficial immunoregulation and immune-mediated neuroprotection. By correlating changes measured in the biological system (e.g. different functions of the T cells or other immune cell subsets) with structural changes of CNS destruction or repair (measured by neuroimaging modalities), and with clinical outcomes, we can understand which biological processes are beneficial and which are harmful in the MS pathogenesis. Additionally, understanding which effects of applied therapies underlie their therapeutic benefit will allow us to define biomarkers that are indicative, and ideally also predictive of the full therapeutic response. We are currently studying immunomodulatory properties of three therapeutic agents: daclizumab, idebenone and rituximab in 3 investigator-initiated Phase I/II clinical trials performed under Investigational New Drug (IND) application (PI, Bibiana Bielekova). Our results are briefly summarized below: Daclizumab is a humanized monoclonal Ab against CD25, which is the alpha chain of the IL-2 receptor (IL-2R). CD25 is highly upregulated on activated T cells and contribution of CD25 to high-affinity IL-2 signaling was believed to be paramount for expansion of effector T cells. Thus, it was expected that daclizumab therapy will result in inhibition of T cell functions. Previously we have demonstrated that daclizumab is highly effective in suppressing MS-related inflammatory activity. However, this therapeutic effect was not paralleled by inhibition of T cells. Instead, we determined that daclizumab selectively expands CD56bright natural killer (NK) cells and that this human-specific NK cell population has immunoregulatory function through its ability to kill activated autologous T cells. The expansion of CD56bright NK cells correlated with treatment outcome, suggestive that it can be utilized as biomarker of therapeutic response. This observation has now been fully reproduced in double-blind, placebo-controlled multicenter Phase II trial of daclizumab in MS. We continue to study the mechanism of action (MOA) of daclizumab in MS in new Phase I open label therapeutic trial of DAC-HYP (second generation of daclizumab) in patients with high-inflammatory MS (protocol 10-N-0125), with the goal to determine all effects of daclizumab on human immune system. We determined that daclizumab expands CD56bright NK cells paradoxically in IL-2 dependent manner;because daclizumab prevents consumption of IL-2 by activated T cells, the excess of IL-2 can be utilized by CD56bright NK cells through intermediate-affinity IL-2 receptors. Furthermore, we determined the mechanism of killing of activated T cells by CD56bright NK cells and the paper describing our novel findings is currently in review. We also determined that daclizumab has effects on other cells of the innate immune system, specifically on mature dendritic cells. The paper describing this MOA is also currently in review. Idebenone is a lipid soluble synthetic analogue of coenzyme-Q (CoQ). As such, it both improves mitochondrial metabolism and has an antioxidant effect. Primary-progressive MS (PP-MS) is resistant to therapy by immunomodulatory agents. Mitochondrial dysfunction coupled with oxidative stress has been proposed as one of the top alternative hypotheses underlying pathophysiology of PP-MS. Therefore, we initiated double-blind phase I/II clinical trial of idebenone versus placebo (protocol 09-N-0197) to test the hypothesis whether mitochondrial dysfunction and oxidative stress contribute to development of neurological disability in PP-MS. Efficacy of idebenone in suppressing oxidative stress intrathecally and its unexplored immunomodulatory effects are being measured by ex-vivo analysis of PBMC functions and CSF biomarkers in enrolled patients. Rituximab is a B cell depleting chimeric monoclonal Ab and has inhibitory effect on MS disease activity in patients with evidence of blood-brain barrier (BBB) breakdown. The current leading hypothesis is that B cells collaborate with antigen-specific T cells and support T cell activation through antigen-presentation functions and mutual co-stimulation. Furthermore, there is evidence that in secondary-progressive MS (SP-MS) patients B cells and T cells form tertiary lymphoid follicles deep within the brain sulci. These ectopic lymphoid follicles recapitulate activation of antigen-specific T and B cells within the intrathecal compartment. Unfortunately, because majority of progressive MS patients do not have overt BBB disruption, only about 0.1% of systemically administered rituximab gains access to CNS compartment, which is not sufficient to deplete CNS B cells. Therefore, we are testing the hypothesis that intrathecal administration of rituximab in patients with SP-MS will effectively deplete B cells in the CNS compartment and this will translate into therapeutic effect of rituximab on biomarkers of CNS tissue injury in the placebo-controlled, Phase II trial of rituximab in low-inflammatory SP-MS (protocol T-N-1235). While there are many agents that have biologically plausible MOA for testing their efficacy in progressive MS, the drug development is currently hampered by the fact that we have no sensitive interim outcome measure. Therefore, current Phase II studies in progressive MS require enrollment of hundreds of MS patients and are prohibitively expensive. Both of our double-blind placebo-controlled investigator-initiated Phase II clinical trials (i.e. idebenone and IT rituximab) share identical adaptive trial design focusing on the evaluation of different (mostly novel) quantitative clinical, functional and imaging outcome measures. The hope is that collected data from these 2 studies will help to define new, more effective clinical trial design for future Phase II therapeutic trials in progressive MS.